👤 Hugh C Hemmings

Major depressive disorder is associated with deficits in hippocampal synaptic plasticity that depend on brain-derived neurotrophic factor (BDNF) release from both axonal and dendritic compartments. Antidepressant efficacy requires enhanced BDNF signaling, thought to be mediated by drug-induced BDNF release from postsynaptic dendritic spines. Here, we show that fast-acting antidepressants rapidly trigger BDNF secretion from presynaptic terminals in hippocampal area CA3. At antidepressant-relevant concentrations, ketamine and its metabolite (2R,6R)-hydroxynorketamine (HNK) induced BDNF release within minutes from mossy fiber terminals of dentate granule neurons in rat hippocampal cultures, with no detectable secretion from dendritic spines. This antidepressant-evoked BDNF release required presynaptic NMDA receptors (preNMDARs). Conditional genetic deletion of preNMDARs from granule neurons abolished ketamine- and HNK-induced BDNF exocytosis in acute mouse hippocampal slices, establishing a presynaptic receptor mechanism for antidepressant-induced neurotrophin release. In CA3 pyramidal neurons that receive mossy fiber input, both compounds induced rapid remodeling of dendritic spines, resulting in increased spine density. Together, these findings identify presynaptic terminals as a previously unrecognized source of antidepressant-evoked BDNF release and establish a new cellular mechanism for the rapid synaptic effects of fast-acting antidepressants. Show less

Indole-3-carbinol (I3C) is a metabolic derivative of glucobrassicin found in cruciferous vegetables. Known for its anticarcinogenic properties, I3C has been shown to target the NEDD4 family HECT E3 ligases, NEDD4-1 and WWP1, yet Show less

The HECT E3 ubiquitin ligases 1 (WWP1) and 2 (WWP2) are responsible for the ubiquitin-mediated degradation of key tumour suppressor proteins and are dysregulated in various cancers and diseases. Here we expand their limited inhibitor space by identification of NSC-217913 displaying a WWP1 IC Show less

We have screened small molecule libraries specifically for inhibitors that target WWP2, an E3 ubiquitin ligase associated with tumour outgrowth and spread. Selected hits demonstrated dose-dependent WWP2 inhibition, low micromolar IC50 values, and inhibition of PTEN substrate-specific ubiquitination. Binding to WWP2 was confirmed by ligand-based NMR spectroscopy. Furthermore, we used a combination of STD NMR, the recently developed DEEP-STD NMR approach, and docking calculations, to propose for the first time an NMR-validated 3D molecular model of a WWP2-inhibitor complex. These first generation WWP2 inhibitors provide a molecular framework for informing organic synthetic approaches to improve activity and selectivity. Show less

The WWP2 E3 ubiquitin ligase has previously been shown to regulate TGFβ/Smad signalling activity linked to epithelial-mesenchymal transition (EMT). Whilst inhibitory I-Smad7 was found to be the preferred substrate for full-length WWP2-FL and a WWP2-C isoform, WWP2-FL also formed a stable complex with an N-terminal WWP2 isoform (WWP2-N) in the absence of TGFβ, and rapidly stimulated activating Smad2/3 turnover. Here, using stable knockdown experiments we show that specific depletion of individual WWP2 isoforms impacts differentially on Smad protein levels, and in WWP2-N knockdown cells we unexpectedly find spontaneous expression of the EMT marker vimentin. Re-introduction of WWP2-N into WWP2-N knockout cells also repressed TGFβ-induced vimentin expression. In support of the unique role for WWP2-N in regulating TGFβ/Smad functional activity, we then show that a novel V717M-WWP2 mutant in the MZ7-mel melanoma cell line forms a stable complex with the WWP2-N isoform and promotes EMT by stabilizing Smad3 protein levels. Finally, we report the first analysis of WWP2 expression in cancer cDNA panel arrays using WWP2 isoform-specific probes and identify unique patterns of WWP2 isoform abundance associated with early/advanced disease stages. WWP2-N is significantly downregulated in stage IIIC melanoma and up-regulated in stage II/III prostate cancer, and we also find isolated examples of WWP2-FL and WWP2-C overexpression in early-stage breast cancer. Together, these data suggest that individual WWP2 isoforms, and particularly WWP2-N, could play central roles in tumourigenesis linked to aberrant TGFβ-dependent signalling function, and also have potential as both prognostic markers and molecular therapeutic targets. Show less